Null mutation of myeloperoxidase in mice prevents mechanical activation of neutrophil lysis of muscle cell membranes in vitro and in vivo

Abstract

Membrane lysis is a common and early defect in muscles experiencing acute injuries or inflammation. Although increased mechanical loading of muscles can induce inflammation and membrane lysis, whether mechanical loads applied to muscle can promote the activation and cytolytic capacity of inflammatory cells and thereby increase muscle damage is unknown. We tested whether mechanical loads applied to mouse muscle cells in vitro can increase membrane lysis, and whether neutrophil-mediated lysis of muscle cells is promoted by mechanical loads applied in vitro and in vivo. Cyclic loads applied to muscle cells for 24 h in vitro produced little muscle cell lysis. Similarly, the addition of neutrophils to muscle cell cultures in the presence of superoxide dismutase (SOD) produced little muscle cell lysis. However, when cyclic mechanical loads were applied to neutrophil-muscle co-cultures in the presence of SOD, there was a synergistic effect on muscle cell lysis, suggesting that mechanical loading activates neutrophil cytotoxicity. However, application of mechanical loads to co-cultures of muscle cells and neutrophils that are null mutants for myeloperoxidase (MPO) showed no mechanical activation of neutrophil cytotoxicity. This indicates that loading promotes neutrophil cytotoxicity via MPO. Activity assays confirmed that mechanical loading of neutrophil-muscle co-cultures significantly increased MPO activity. We further tested whether muscle membrane lysis in vivo was mediated by neutrophils when muscle was subjected to modified loading by using a mouse model of muscle reloading following a period of unloading. We observed that MPO-/-soleus muscles showed a significant 52% reduction in membrane lysis compared to wild-type mice, although the mutation did not decrease inflammatory cell extravasation. Together, these in vitro and in vivo findings show that mechanical loading activates neutrophil-mediated lysis of muscle cells through an MPO-dependent pathway.

Figure 1. Cytolysis of wild-type mouse myotubes in lactate dehydrogenase (LDH) release cytotoxicity assays

A, muscle cytolysis in the absence (Muscle only) or presence (Muscle + Neutrophils) of wild-type neutrophils. Exogenous superoxide dismutase (SOD) was added to some preparations. All treatments used 5000 neutrophils mm⁻². Data from cultures that were not subjected to mechanical loading are shown by black columns. Data from cultures subjected to mechanical loading are shown by grey columns. B, identical treatments as shown in A, but neutrophils from myeloperoxidase (MPO) −/− mice were used in the assays. * Significantly different from corresponding non-loaded treatment. # Significantly different from muscle cells without neutrophils treated under otherwise identical conditions. P < 0.05. n = 6 replicates per treatment. Bars represent s.e.m.

Figure 2. MPO activity in supernatants of muscle cells or muscle cells with neutrophil co-cultures

Conditions were identical to those used in Fig. 1A except that all treatments used 25 000 neutrophils mm⁻². * Significantly different from corresponding non-loaded treatment. # Significantly different from muscle cells without neutrophils treated under otherwise identical conditions. P < 0.05. n = 6 per treatment. Bars represent s.e.m.

Figure 3. MPO activity of neutrophil cultures in the absence of muscle cells

Cultures were either subjected to mechanical loading or not loaded. Some preparations were activated with phorbol 12-myristate 13-acetate (PMA). Mechanical loading did not significantly increase MPO activity in the supernatant of any treatment compared to identically treated samples in the absence of mechanical loading. * Significantly different from wild-type neutrophils subjected to identical experimental treatments. P < 0.05. n = 6 per treatment. Bars represent s.e.m. Data sets without apparent error bars had errors that were too small to appear at the scale of this graph.

Figure 4. Transfer of conditioned media from muscle cells subjected to loading or not subjected to loading does not increase MPO activity in neutrophil cultures

Medium was collected from muscle cultures after 24 h of cyclic loading or non-loading, transferred to neutrophil cultures and then MPO activity measured after 24 h. Neutrophil cultures contained 5 × 10⁶ neutrophils ml⁻¹. ‘% Conditioned Media’ indicates the percentage of the total media in the cultures that consisted of conditioned media. There was no significant difference in MPO activity between cultures receiving conditioned media from loaded or non-loaded muscle cell cultures at any concentration of conditioned media. Bars represent s.e.m.n = 6.

Figure 5. Null mutation of MPO does not reduce myeloid cell invasion of reloaded muscle

Soleus muscles from ambulatory control mice (Amb), mice subjected to 10 days of hindlimb unloading only (Unload) or unloading followed by 24 h reloading (Reload) were assayed for neutrophil concentration (A) and macrophage concentration (B). # Significantly different from ambulatory mice of the same genotype. No significant differences were found between MPO −/− and wild-type mice in any experimental group. Bars represent s.e.m.n = 6.

Figure 6. Soleus muscle fibre injury assessed by bright intracellular fluorescence of procion orange

Treatment groups are the same as for Fig. 5. * Significantly different from ambulatory mice of the same genotype. # Significantly different from MPO −/− mice in the same experimental group. Bars represent s.e.m.n = 6.

Figure 7. Frequency distributions of muscle fibres over the range of intensities of intracellular fluorescence of procion orange

Intracellular fluorescence was measured in individual fibres in entire cross-sections of soleus muscles from each treatment group (n = 6 per group). A total of more than 34 000 individual fibres was analysed. A rightward shift of peaks on the abscissa indicates an increase in the frequency of fibres with membrane lesions. Frequency distribution of intracellular fluorescence for wild-type (dashed line) or MPO −/− (continuous line) muscle fibres of ambulatory control (A), unloaded (B) or reloaded (C) soleus muscle fibres.

Figure 8. Schematic diagram of free radical production that may be influenced by mechanical loading of neutrophils in skeletal muscle

Black boxes indicate substrates or products that are benign or have low toxicity. Red boxes indicate substrates or products that are cytotoxic. Red lettering indicates enzymes that are activated by mechanical loading. Green lettering indicates enzymes that are inhibited by mechanical loading.